Note
EXPRESSION OF THE SIGMA
35AND CRY2AB GENES
INVOLVED IN Bacillus thuringiensis VIRULENCE
Ana Maria Guidelli-Thuler1; Irlan Leite de Abreu1; Manoel Victor Franco Lemos2*
1
UNESP/FCAV - Programa de Pós-Graduação em Microbiologia Agropecuária.
2
UNESP/FCAV- Depto. de Biologia Aplicada à Agropecuária - Via de Acesso Prof. Paulo Donato Castellane, s/n - 14884-900 - Jaboticabal, SP - Brasil.
*Corresponding author <mvictor@fcav.unesp.br>
ABSTRACT: There are several genes involved in Bacillus thuringiensis sporulation. The regulation and expression of these genes results in an upregulation in Cry protein production, and this is responsible for the death of insect larvae infected by Bacillus thuringiensis.Gene expression was monitored in Bacillus thuringiensis during three developmental phases. DNA macroarrays were constructed for selected genes whose sequences are available in the GenBank database. These genes were hybridized to cDNA sequences from B. thuringiensis var. kurstaki HD-1. cDNA probes were synthesized by reverse transcription from B. thuringiensis RNA templates extracted during the exponential (log) growth, stationary and sporulation phases, and labeled with 33PadCTP. Two genes were differentially expressed levels during the different developmental phases. One of these genes is related to sigma factor (sigma35), and the other is a cry gene (cry2Ab). There were differences
between the differential levels of expression of various genes and among the expression detected for different combinations of the sigma factor and cry2Ab genes. The maximum difference in expression was observed for the gene encoding sigma35 factor in the log phase, which was
also expressed at a high level during the sporulation phase. The cry2Ab gene was only expressed at a high level in the log phase, but at very low levels in the other phases when compared to the sigma35.
Key words: cry genes, gene expression, sigma factor
EXPRESSÃO DOS GENES SIGMA
35E
CRY2AB
ENVOLVIDOS NA
VIRULÊNCIA DE
Bacillus thuringiensis
RESUMO: Muitos genes estão envolvidos nos mecanismos de esporulação da bactéria Bacillus thuringiensis. A regulação e expressão desses genes resultam em uma produção massiva da proteína Cry, responsável pela morte das larvas de muitos insetos.Neste trabalho monitorou-se a expressão de genes de Bacillus thuringiensis, ao longo de três fases de seu desenvolvimento. Foram construídos macroarrays de DNA dos genes selecionados, cujas seqüências estão disponibilizadas no GenBank. Estes genes foram hibridizados com cDNAs obtidos de B. thuringiensis kurstaki HD-1. As sondas de cDNA foram sintetizadas a partir da transcrição reversa do RNA da bactéria, extraído durante as fases de crescimento logarítmico, estacionária e esporulativa, marcadas com 33PadCTP. A expressão diferencial encontrada foi significativa para dois genes de B.thuringiensis, um relacionado aos fatores sigma (sigma35) e outro ao gene cry (cry2Ab). Detectaram-se diferenças entre as médias de expressão do fator
sigma e do gene cry2Ab. Os valores máximos de expressão diferencial foram obtidos para o gene codificador do fator sigma35 na fase log e na fase esporulativa. Na análise de médias observou-se
expressão do gene cry2Ab apenas na fase log; no entanto, de forma bem mais baixa quando comparado com a expressão de sigma35, nas três fases.
Palavras-chave: genes cry, expressão gênica, fator sigma
INTRODUCTION
Many genes are involved in sporulation mecha-nisms. It is estimated that about 800 genes are active in this phase in Bacillus subtilis, and this is in
During sporulation, the regulation of cry genes results in upregulation of the Cry proteins, which kill insect larvae (Lereclus et al., 2000). These proteins also allow the germination of bacterial spores and multipli-cation of bacterial cells in larval intestines. The pro-duction of extracellular factors in response to nutrient deficiency allow bacterial cells to wound and invade host tissues, accessing alternative nutrient sources and providing favorable conditions for a new multiplica-tion or exponential growth cycle.
The expression of cry genes is regulated by two mechanisms. One of them, which regulates most of
the cry genes, depends on specific sigma factors of
the sporulation phase. The other mechanism does not depend on sporulation, and regulates factors that are typical of the stationary phase, e.g., cry 3 (Valadares-Inglis et al., 1998).
The selection of B. thuringiensis isolates with high virulence is a very important step for the devel-opment of biopesticides (Polanczyk & Alves, 2005).
There have been some reports that characterized B.
thuringiensis according to cry gene content (Bravo et al., 1998; Ferrandis et al., 1999; Uribe et al., 2003, Vilas-Bôas & Lemos, 2004), but the expression level of these genes, or even their product precursors, have still not been completely elucidated. Therefore, the search for the phases in which the cry genes and their precursors are expressed, as well as their expression levels in a standard strain such as HD-1, becomes of
fundamental importance for the selection of other B.
thuringiensis strains that may be used for insect con-trol.
In this study, the differential expression
of genes from B. thuringiensiskurstaki HD-1 related
to virulence, sporulation and crystal protein forma-tion was monitored in order to determine the genes expressed at the logarithmic, stationary and sporula-tion phases of the development cycle of this bacte-rium.
MATERIAL AND METHODS
Bacterial strain - The B. thuringiensis var. kurstaki
HD-1 strain used was obtained from the ‘Bacillus
Ge-netic Stock Center’ and is kept at the Laboratory of Bacterial Genetics and Applied Biotechnology, FCAV -UNESP/ Jaboticabal, São Paulo, Brazil.
Gene selection for DNA array production - For DNA array production, B. thuringiensis genes were selected, cloned, sequenced and annotated based on informa-tion from the GenBank database. The genes selected (Table 1) are related to virulence factors (expressed during the stationary phase), sporulation mechanisms
and formation of protein crystals (expressed during the stationary and sporulation phases). DNA arrays were produced at the ‘Brazilian Clone Collection Center’ (BCCCenter).
Extraction and quantification of genomic DNA - B. thuringiensis var. kurstaki (HD1) cells weregrown in
a semi-solid medium (Nutrient Agar) (Gordon et al.,
1973), from ‘BIOBRÁS’ and incubated at 30ºC for approximately 18 h. Colonies isolated from each line were inoculated in 50 mL of Brain Heart Infusion me-dium, also from ‘BIOBRÁS’ and were grown with agi-tation at 200 rpm at 30ºC for 4 h and 30 min.
Genomic DNA was extracted as described by Marmur (1961), and after ethanol precipitation, the DNA samples were resuspended in TE buffer (10 mM Tris; EDTA 1 mM, pH 8.0) and stored at –20ºC. DNA was quantified from samples of each strain using a spectrophotometer (BECKMAN, model DU-640B), and DNA quality was determined by electrophoresis in a 0.8% agarose gel with TBE buffer (89 mM Tris; 89 mM boric acid and 2.5 mM EDTA, pH 8.2), at 90 V for 90 min. The DNA samples were diluted to 10 ng
µL–1 for analysis.
Design of oligonucleotide primers - Pairs of oli-gonucleotide primers were designed using the
Gene Runner 3.0 software (Hastings Software, Inc.) and sequences obtained from GenBank (Table 1). The selected genes were amplified by PCR (DeRisi et al., 1997) using genomic DNA as a template, and the PCR products were designed to be used in macroarrays for the evaluation of gene expression at different developmental stages of B. thuringiensis var. kurstaki (HD1).
Oligonucle-otides were synthesized by MWG (The Genomic
Com-pany) and primers were used at a concentration of
1 pmol mL–1 in the PCRs.
B. thuringiensis var. kurstaki HD-1 DNAwas amplified using the specific primers. PCR amplifica-tion reacamplifica-tions were carried out in a final volume of
20 µL in aPTC – 100TM
thermal cycler (MJ Research).
Amplifications with the primers for cry1Ac, spo0I,
ori44, sigma 28 (K), slp, cry11A and chi were
per-formed with 0.6 mM MgCl2 (Invitrogen), 250 µM
dNTPs, 1 pmol primers, 1X PCR buffer (Invitrogen), 1U Taq polymerase (Invitrogen) and 30 ng genomic
DNA. Amplifications with the primers cry1Ab,
cry1-2, cry1Ia, pew3, gyrB, cry3Ca, cry1Aa, cry218,
crybns3, Hkna, npra, ori43, cry2Ab, sigma35 (K) and (I), mob1, cry10Aa, cyt2Ba, sigma 28(I), cry4B, Bt8,
spoIIID and cry4Aa were performed using the same
conditions, except that the MgCl2 concentration was
Table 1 - Oligonucleotide primers used in this study.
Primers Sequences Base pairs amplified
Cry1Aa D17518
5' ATATTTCCTTGTCGCTAACGC 3' 5' CTGTTATTTGATGCCCTGACC 3'
850
Cry1Ab M12661
5' CGGGATTAGAGCGTGTATG 3' 5' CATCCAGCGAATCTACCG 3'
450
Cry1Ac U87793
5' ATCGCTCGTCTATCGGCATTG 3' 5' AGCCAGCCCTCACGTTCTTC 3'
400
Cry218 AJ002514
5' ACAGTGCCCTTACAACCG 3' 5' CCATAAGCAAATTCTGTCCC 3'
403
Cry1B M23723
5' TACAGATACCCTTGCTCGTG 3' 5' GGTGTTGATAAAGGAGGGAG 3'
798
Cry1-2 D00117
5' GCTCAGGGCATAGAAGGAAG 3' 5' TTCTGTGGCGGTATTTCATC 3'
600
CryV465 U07642
5'CTATGGCAGAGAACGAAG 3' 5'CAGACTTGAGAGGATTAGG 3'
650
Crybns3 Y09663
5' TGGTCAGGGCATCAAATAAC 3' 5' GCTCTCAAGGTGTAAACTGC 3'
401
Hkna U03552
5' ATGCTACCCGCCACTTCAG 3' 5' CCCTCCATTTCCATACGAC 3'
422
Npra L77763
5' ATCTAACTGAAGCAACTGGC 3' 5' CTTTACCGCTGTCTGCCTC 3'
495
Ori43 M60513
5' CCCGCATCATCCATCATAG 3' 5' TTGTCCCGTAGAAAGTTGC 3'
455
Ori44 M60465
5' TCGTAAAGCTGGAAAGAAAGG 3' 5' AAATACCAAGCATCTCTGTCG 3'
402
Pew3 E01234
5' GCTCAGGGCATAGAAAGAAG 3' 5' TCTGTGGCGGTATTTCATC 3'
400
Spo0A X80639
5' GAATGTGTTGCGAAAGGG 3' 5' TCCATCTACTGTTGTTGCTG 3'
600
Slp X62090
5' ACTTGCTCGTTAGGTTGCTC 3' 5' AGCAAGGAAGAGAAGTAGTCG 3'
543
Cry4B X07423
5' ATCTCGTGTAAGTGCTGCCTC 3' 5' AAGAGAGCCTAATAACCAGTCC 3'
541
Cry11A M31737
5'GGATGGATAGGAAACGGAAG 3' 5' ATACTGCCGTCTGTTGCTTG 3'
593
MOB1 U67921
5' CACAATGATGAGGCAAGTC 3'
5'TTGGGCTTTACCTCCGTC 3' 404
Bt8 X07423
5' TCCGTTAGCGAATGACTTAC 3' 5' AAATAAACTGCGGTCTCTCG 3'
477
Cry10Aa M12662
5' CATCCTGCTACCGAAACG 3' 5' ATCAAATCGCCTCCTATCC 3'
400
SpoIIID D28169
5' GACAAGAAAGACAGTGCGTG 3' 5' CCCACCATACTTCTCGCTTC 3'
538
Cry4Aa Y00423
5' GTGTCCAATCTGAACTTACTCG 3' 5' GGTTTCCTTGATTCTCTCTTCG 3´
501
Cry2Ab X55416
5' ACGCCATTTGTTAGGAGTTG 3' 5' TTTCCATTCCCTCTAAGCG 3'
512
Cyt2Ba U52043
5' CAAATGGTCTTCCTAATGCAG 3' 5' TATGATTTGGACGATGTAAGC 3'
498
Gyrb AF136390
5' TGGTGGGAAGTTTGACGG 3' 5' TCACGAACATCCTCACCAG 3'
654
Rep63 AJ011655
5' GCGTAAGCCCATTCTCTATTC 3' 5' CAGGTGTTGTGTTGGATGAAAG 3'
749
Sigma 28 X56696
5' CGCAGCCGTTATCATCAG 3' 5' CCCTTACAAACTCGTGGAAC 3'
606
Sigma 35 X56697
5' TGGGATGGGAATGAACTG 3' 5' TCTCGTCAAAGTGTTCCCTC 3'
451
Cry3Ca X59797
5' TGACAAGAAAGGGAGGAAG 3' 5' TTCGTCCATCTCGTAAAGTC 3'
501
chi AF526379
5' GCAGATTCACCAAAGCAAAG 3' 5' TCCCAGTCTAAATCTACGCC 3'
Genomic DNA amplification and analysis of
ampli-fied products - After sample preparation,
thermocycling was carried out using the following con-ditions: one initial denaturing step of 2 min at 94ºC, 30 cycles (1 min at 94ºC; 1 min at 48ºC; 1 min at 72ºC) and one final extension step at 72ºC for 10 min, with the primers mob1, cry10Aa, cyt2Ba, sigma 28(I), sigma35 (K) and (I), cry1Aa, cry218, crybns3, Hkna,
npra, ori43 and cry2Ab. A similar program was used for the other primer sets, but the annealing tempera-ture was changed to 50ºC.
Amplified products were analyzed by EBAGE (ethidium bromide gel electrophoresis) using a 1.5% gel (Sambrook & Russel, 2001) and TBE buffer (70 V for 2.5 h). DNA fragments were observed under UV light and gels were visualized using the Gel Documen-tation System (BIO-RAD).
The amplification products were then
resus-pended in 50% DMSO at concentrations of 2.5 µg
µL–1
and 100 µg µL–1
, and transferred to a 384-well plate, then applied to nylon membranes at the Brazil-ian Clone Collection Center (BCCCenter).
Bacterial culture for total RNA extraction
-Samples were extracted for RNA preparation at regu-lar intervals during bacterial culture (Gordon et al., 1973). B. thuringiensis var. kurstaki HD-1 RNA at veg-etative growth, stationary and sporulation phases was extracted (Ribier & Lecadet, 1973; Bulla Jr, et al., 1980).
Growth phases were defined through succes-sive microscopic analyses of the cultures and by spec-trophotometer readings at 600 nm.
The strain was cultured in Nutrient Agar
(BIOBRÁS) (Gordon et al., 1973) and incubated at
30ºC for approximately 18 h. Colonies isolated from the strains were inoculated in 50 mL of nutritious broth medium (BIOBRÁS) and cultured with shaking at 200 rpm at 30ºC for 12 h (DO 0.4) to prepare the pre-inoculum. Inocula for the three growth phases were obtained by culturing 1 mL of the pre-inoculum in 50 mL of medium. Cells were cultured for 4h30 min (DO 0.1), 6h30 min (DO 0.2) and 9 h (DO 0.3) to obtain log phase, stationary phase and sporulation phase samples, respectively.
Samples were collected at each of the three phases of development and total RNA was immediately extracted.
Total RNA extraction - Bacterial cultures (50 mL) were centrifuged at 4500 g for 10 min at 4ºC, and to-tal RNA extraction was performed as described by Cabanes et al. (2000). After extraction, RNA samples were quickly dried and resuspended in DEPC-treated water (0.01%) and stored at –80ºC after
quantifica-tion. RNA samples were analyzed by
spectrophotom-etry (BECKMAN, model DU-640B) (Sambrook &
Russel, 2001), and RNA integrity was analyzed by electrophoresis in a 1.2% agarose gel containing 6.7% formaldehyde, according to Soares & Bonaldo (1997).
Production of cDNA probes for macroarray hybrid-ization - Complementary DNA was synthesized by
re-verse transcription of the RNA extracted from B.
thuringiensis var. kurstaki HD-1 samples from differ-ent developmdiffer-ental phases.
Thirty micrograms of total RNA (in 6 µL) was
added to 156 pmols of Random Primer Pd(N)6
(Amersham Pharmacia Biotec Inc.). Samples were in-cubated at 75ºC for 10 min to allow primers to an-neal, and then transferred to ice bath. Five microliters
of First Strand Buffer (5X, BRL) (Invitrogen), 2.5 µL
of DTT (100 mM), 2.0 µL of RNAguard (Invitrogen),
2.5 µL of ATG (10 mM each) and 5 µL of 33
P αdCTP
(50 µCi) were then added. The mix was then heated
to 42ºC and 1.25 µL of Superscript II (Invitrogen, 200 U µL–1
) was added. Samples were gently stirred and
incubated at 37ºC for 20 min. Then, 1.25 µL of dCTP
(10 mM) was added to the mix and the mix was in-cubated at 37ºC for approximately 2 h. Samples were then denatured at 94ºC for 5 min and incubated at 37ºC
for 15 min after adding 1.4 µL NaOH (5M). After
in-cubation, 1.8 µL HCl (3.94M) and 7.0 µL of Tris-HCl
(1M) pH 7.5 were added. The total volume of the probe was measured in a scintillator and purified us-ing a G-50 column (Amersham Pharmacia Biotec Inc.). After purification, the probe volume was mea-sured again. The incorporation efficiency of purified probe should range from 30 to 50%, compared to the unpurified version.
Macroarray hybridization
Pre-hybridization - Membranes were pre-hy-bridized according to a Southern protocol (Sambrook & Russel, 2001) for 4 h at 42ºC. Hybridization cylin-ders containing 10 ml of solution were used, and each cylinder contained only one membrane.
Hybridization - The hybridization solution was prepared as recommended for a Southern protocol (Sambrook & Russel, 2001) and kept at 42ºC until use. After purification and denaturing, the probe was added to a 50 mL tube containing the hybridization solution for 42 h at 42ºC.
Detection and measurement of hybridiza-tion signals - Hybridization signals were obtained from 30 genes from the three development phases of this bacterium (treatments). Each gene was spotted three times onto each membrane, and two different mem-branes were used, with a completely randomized de-sign, and therefore there were six repetitions of each treatment.
After the membranes were exposed, the phosphoimaging screens were analyzed with a Cyclone Phosphor-Imager (Packard Instruments), and hybrid-ization signals were measured using the Optiquant soft-ware, and expressed in DLU (Digital Light Units).
The probe hybridization data were normalized by subtracting background-related values from regions of the image that did not correspond to DNA spots. The genes expression levels were subject to analysis of vari-ance (ANOVA), being their means compared by a Tukey
test (p ≤ 0.05). These analyses were performed with
the SAS statistical program (SAS Institute, 1995).
RESULTS AND DISCUSSION
Gene expression analysis - PCR products were ana-lyzed by electrophoresis in an agarose gel in order to determine their size and purity. This procedure is con-sidered one of the most important steps in large-scale gene expression studies (Deyholos & Galbraith, 2001). It confirms that a unique amplicon is present and pro-vides an estimate of the concentration of the ampli-fied product.
Three developmental phases of B. thuringiensis
kurstaki HD-1 were analyzed in order to investigate the expression of 30 genes. The sequences of these genes can be found in GenBank, which also includes recent citations of other sequences obtained by well-known researchers, allowing specific primers to be designed for the detection of these genes. The genes that were chosen for the present study play important biological roles in this bacterium and encode the Cry proteins. These proteins mediate highly specific toxic activities, allowing larvae from insect pest species to be controlled by this bacterium. The other genes evaluated in this study are involved in basic metabolic processes, and act to initiate the process that ultimately leads to cry1 gene expression. Our results indicate that two important genes related to physiological processes that take place before sporulation are differentially expressed at phases in bacterial development.
Two B. thuringiensis genes exhibited signifi-cant variations in their levels of expression (Figure 1);
one gene was related to sigma factors (sigma35), and
the other was related to the cry gene (cry2Ab). The
sigma factor is expressed during all three phases
ex-amined, laying an important role in transcription. RNA polymerase begins transcription by recognizing a spe-cific region of DNA, the promoter. Promoter recog-nition by RNA polymerase is mediated by a sigma fac-tor, which attaches to the enzyme, and identifies pro-moter sites. The action of several types of propro-moters and sigma factors regulate cell functions, allowing a balance of physiological activities to be maintained.
Therefore, the observation that sigma35
factor is ex-pressed at all the development stages in this study con-firms its direct involvement in the cry1Aa promoter site
recognition of B. thuringiensis, as reported by Adams
et al. (1991). The sigma35
holoenzyme is also related to other
cry genes, as observed by Tounsi & Jaoua (2002), who
suggested that the transcription of a cry1Ia promoter
in B. thuringiensis var. kurstaki is mediated by this holoenzyme. They described this enzyme as an RNA polymerase specific to the sporulation phase
associ-ated with the BtI promoter. However, the sigma38
–de-pendent BtII is apparently not involved in cry1Ia tran-scription. Despite the fact that both promoters are ac-tive during the medium and late sporulation stages of
B. thuringiensis var. kurstaki HD-1-Dipel, sigma35
was expressed in the sporulation, vegetative and stationary phases in this study, but was expressed at lower lev-els in the stationary phases (Figure 1).
The high expression of the sigma35
factor from the log phase onwards suggests that the activation of
cry genes begins in this phase. This is because these
genes are related to sporulation, which depends on the activation of several genes in cascade, including the sigma35
factor. This hypothesis is also supported by the fact that transcription from the amyE promoter increased substantially as cell growth reached the log phase. The
gene that encodes sigma35
is a significant promoter, when one observes cry genes expression, indicating that the presence of other promoters might change the ex-pression in each phase in which the gene is expressed.
Figure 1 - Hybridization of B. thuringiensis genes with B. thuringienis kurstaki HD-1 cDNAs at three developmental phases. The image was produced by the Cyclone Phosphor-Imager (Packard Instruments). A: sigma35. B: cry2Ab
Phase 1 Phase 2 Phase 3
B
A
B
A
B
Expression of cry1Ac begins in the B. thuringiensis log phase(Chak et al., 1994). cry1Ac ex-pression in B. thuringiensis kurstaki Tt14 is regulated
by the amyE promoter, which is dependent on a
spe-cific sigma factor (sigma43
) (Yang et al., 2003). Other
studies have shown that cry gene expression is
depen-dent on sigma28 or sigma35 factors (Nicholson et al., 1987).
The cry2Ab gene was also expressed in the log
phase in B. thuringiensis kurstaki HD-1, although at
much lower levels than the gene encoding sigma35.
Despite the fact that cry2Ab has been described as
Diptera-specific, it has also been reported to have ef-fects upon Lepidoptera (Schnepf et al., 1998). Its ex-pression in the log phase, together with the gene
en-coding sigma35 factor, suggests that this holoenzyme
is not responsible for recognition of the cry2Ab
pro-moter site. This explains why it was not possible to
detect the expression of the cry1 genes at this stage
of development. The sigma35 factor is responsible for
promoter site recognition; there would be no expres-sion of these genes before expresexpres-sion of the gene
en-coding the sigma factor. In the log phase, the cry2Ab
gene is transcribed using an alternative promoter un-related to sigma35
. This unknown alternative promoter
may also activate cry2Ab expression at earlier
devel-opmental phases, before the cry1A genes are expressed. There was a difference in the levels of
expres-sion of the sigma factor and cry2Ab (Tukey test,
p < 0.01) (Tables 2 and 3). The highest mean of ex-pression was observed in the log phase for the gene
encoding sigma35, and the second highest expression
was during the sporulation phase (Table 3). Cry2Ab
was only expressed at significant levels in the log phase, but the expression of this gene was still low when com-pared with the sigma-encoding gene during all devel-opmental phases examined (Tables 2 and 3).
The expression of cry2Ab is highly relevant
even at low levels, since this gene has previously been studied using transgenic varieties of cotton, maize and
tomato, which simultaneously express cry1Ac and
cry2Ab, the so-called ‘second generation’ of
commer-Table 2 - Variance analysis (ANOVA) for expression genes (DLU) relative to exponential, logarithmic and stationary developmental phases.
Sources of Variation Degrees of Freedom Mean Squares (DLU) F value P value
Experiment (EX) 1 5142374052.00 368.58 < 0.0001
Gene (GN) 1 78220040180.00 5606.42 < 0.0001
Phase (PS) 2 17437460829.00 1249.83 < 0.0001
EX*GN 1 2562660381.00 183.68 < 0.0001
EX*PS 2 19135176304.00 1371.51 < 0.0001
GN*PS 2 13192121728.00 945.54 < 0.0001
EX*GN*PS 2 16906193773.00 1211.75 < 0.0001
cial transgenic genotypes. A combination of cry genes
is being used to minimize selection pressures and to avoid Bt-resistant insect pests. The use of cry2Ab al-lows monitoring programs and preventive resistance control measures to be implemented, because cross-resistance with the cry2Ab and cry1Ac combination is much lower than when using the combination of
cry1Ac and cry1Aa, as observed for the pink bollworm (Tabashnik et al., 2002) and cry1Ac and cry1Aa reported in Heliotis virescens with transgenic Bt maize (Fuentes et al., 2003).
In the log phase, only the gene encoding sigma35
was expressed at high levels. This result con-trasts to the results from a previous study that reported that both cry1Aa and sigma35 are expressed in the log phase (Schnepf et al., 1998). However, this study re-porting simultaneous expression of cry1Aa and sigma35
used an in vivo fusion of the cry1Aa’-‘lacZ genes,
whereas gene expression was analyzed in the natural state without gene fusion in our study, which may ex-plain the differences in the results between these two studies.
In addition to sigma35, we also attempted to
analyze the expression of the gene that encodes sigma28
using a specific set of primers. However, no
sigma28 expression was observed in any of the
devel-opmental phases, thus suggesting that sigma28 is not
involved in the recognition of the cry promoters stud-ied here.
Based on the finding that no other genes were significantly expressed during the developmental stages studied in these experiments, we suggest that new studies should consider the development phases de-fined in this work and even divide them into shorter time periods in order to attempt to explain the lack of expression of genes that are supposedly related to
sporulation and virulence factors in B. thuringiensis
var. kurstaki HD1-Dipel. It is also important to men-tion that the absence of expression of other genes us-ing macroarray techniques may be simply due to the lack of information about the time intervals between
subtilis, seven stages have been defined during sporu-lation, and sporulation is mediated by activation of genes through a signaling cascade. About 800 genes are involved in sporulation in B. subtilis and other spe-cies from this genus (Mandelstam, 1969).
CONCLUSION
During the log developmental phase of the B.
thuringiensis kurstaki-HD-1 bacterium, the genes en-coding sigma35
and cry2Ab were expressed at the high-est levels.
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Received February 06, 2007 Accepted October 06, 2008
Table 3 - Comparison between sigma35 and cry2AB gene
expression means at exponential, logarithmic and stationary developmental phases.
Means followed by different letters in the column are different (Tukey test, p < 0.01).
Genes Phases Means
sigma35 Phase 1 (log) 95443.33 a
sigma35 Phase 2 (stationary) 13608.47 c
